Classical Identification of Bacteria
Bacteria are classically identified by their ability to utilize different substrates as a source of carbon and nitrogen through the use of biochemical tests such as the API20E.TM. system (bioMerieux). Susceptibility testing of gram-negative bacilli has progressed to microdilution tests. Although the API and the microdilution systems are cost-effective, at least two days are required to obtain preliminary results due to the necessity of two successive overnight incubations to isolate and identify the bacteria from the clinical specimen. Some faster detection methods with sophisticated and expensive apparatus have been developed. For example, the fastest identification system, the autoSCAN-Walk-Away.TM. system (Dade Diagnostics) identifies both gram-negative and gram-positive bacterial species from isolated colonies in as little as 2 hours and gives susceptibility patterns to antibiotics in only 7 hours. However, this system has an unacceptable margin of error, especially with bacterial species other than Enterobacteriaceae (Croize J., 1995, La Lettre de l'Infectiologue 10:109-113; York et al., 1992. J. Clin. Microbiol. 30:2903-2910). Nevertheless, even this fastest method requires primary isolation of the bacteria as a pure culture, a process which takes at least 18 hours for a pure culture or 2 days for a mixed culture.
Urine Specimens
A large proportion (40-50%) of specimens received in routine diagnostic microbiology laboratories for bacterial identification are urine specimens (Pezzlo, 1988, Clin. Microbiol. Rev. 1:268-280). Urinary tract infections (UTI) are extremely common (affect up to 20% of women) and account for extensive morbidity and increased mortality among hospitalized patients (Johnson and Stamm, 1989, Ann. Intern. Med. 111:906-917). UTI are usually of bacterial etiology and require antimicrobial therapy. The gram-negative bacillus Escherichia coli is by far the most prevalent urinary pathogen and accounts for 50 to 60% of UTI (Pezzlo, 1988, Clin. Microbiol. Rev. 1:268-280). The prevalence for bacterial pathogens isolated from urine specimens observed recently at the "Centre Hospitalier de l'Universite Laval (CHUL)" is given in Tables 1 and 2.
Conventional Pathogen Identification from Urine Specimens
The search for pathogens in urine specimens is so preponderant in the routine microbiology laboratory that a myriad of tests have been developed. The gold standard is still the classical semi-quantitative plate culture method in which a calibrated loop of urine is streaked on plates and incubated for 18-24 hours. Colonies are then counted to determine the total number of colony forming units (CFU) per liter of urine. A bacterial UTI is normally associated with a bacterial count of 10.sup.7 CFU/L or more in urine. However, infections with less than 10.sup.7 CFU/L in urine are possible, particularly in patients with a high incidence of diseases or those catheterized (Stark and Maki, 1984, N. Engl. J. Med. 311:560-564). Importantly, close to 80% of urine specimens tested are considered negative (less than 10.sup.7 CFU/L; Table 3).
Accurate and rapid urine screening methods for bacterial pathogens would allow a faster identification of negative specimens and a more efficient clinical investigation of the patient. Several rapid identification methods (Uriscreen.TM., UTIscreen.TM., Flash Track.TM. DNA probes and others) were recently compared to slower standard biochemical methods which are based on culture of the bacterial pathogens. Although much faster, these rapid tests showed low sensitivities and poor specificities as well as a high number of false negative and false positive results (Koenig et al., 1992. J. Clin. Microbiol. 30:342-345; Pezzlo et al., 1992. J. Clin. Microbiol. 30:680-684).
Urine specimens found positive by culture are further characterized using standard biochemical tests to identify the bacterial pathogen and are also tested for susceptibility to antibiotics. The biochemical and susceptibility testing normally require 18-24 hours of incubation.
Any Clinical Specimens
As with urine specimens which were used here as an example to show the need for rapid and accurate diagnostic tests for bacterial detection and identification directly from clinical specimens, our probes and amplification primers are also applicable for bacterial detection and identification directly from any other clinical specimens such as blood cultures, blood, sputum, cerebrospinal fluid and others (Table 4). The DNA-based tests proposed in this invention are superior in terms of both rapidity and accuracy to standard biochemical methods currently used for routine diagnosis from any clinical specimens in Microbiology Laboratories. Clinical specimens from organisms other than humans (e.g. other primates, mammals, farm animals or livestock) may also be used.
A High Percentage of Culture Negative Specimens
Among all the clinical specimens received for routine diagnosis, approximately 80% of urine specimens and even more (around 95%) for other types of clinical specimens are negative for the presence of bacterial pathogen (Table 4). It would therefore be desirable, not only to identify bacterial species, but also to screen out the high proportion of negative clinical specimens by detecting the presence of any bacteria (i.e. universal bacterial detection).
Towards the Development of Rapid DNA-Based Diagnostic Tests
A rapid diagnostic test should have a significant impact on the management of infections. For the identification of pathogens and antibiotic resistance genes in clinical samples, DNA probe and DNA amplification technologies offer several advantages over conventional methods. There is no need for culture of the bacterial pathogens, hence the organisms can be detected directly from clinical samples thereby reducing the time associated with the isolation and identification of pathogens. DNA-based technologies have proven to be extremely useful for specific applications in the clinical microbiology laboratory. For example, kits for the detection of fastidious organisms based on the use of hybridization probes or DNA amplification for the direct detection of pathogens from clinical specimens are commercially available (Tenover F. C., and E. R. Unger. 1993. "Nucleic Acid Probes for Detection and Identification of Infectious Agents", pp. 3-25. In Persing, D. H., T. F. Smith, F. C. Tenover, and T. J. White (ed.) Diagnostic Molecular Microbiology: Principles and Applications. American Society for Microbiology, Washington, D.C.).
Others have developed DNA-based tests for the detection and identification of some of the bacterial pathogens for which we have identified species-specific sequences (PCT patent application Ser. No. WO 93/03186). However, their strategy was based on the amplification of the highly conserved 16S rRNA gene followed by hybridization with internal species-specific oligonucleotides. The strategy from the present invention is different, simpler and more rapid because it allows the direct amplification of species-specific, genus-specific or universal bacterial targets using oligonucleotides derived from genomic DNA fragments other than the 16S rRNA genes or from antibiotic resistance DNA sequences which are derived either from the genome or from extrachromosomal elements.
Although there are diagnostic kits or methods already used in clinical microbiology laboratories, there is still a need for an advantageous alternative to the conventional culture identification methods to improve the accuracy and the speed of the diagnosis of bacterial infections. Besides being much faster, DNA-based diagnostic tests are more accurate than standard biochemical tests presently used for diagnosis because the bacterial genotype (e.g. DNA level) is more stable than the bacterial phenotype (e.g. biochemical properties).
Knowledge on the genomic sequences of bacterial species continuously increases as verified from the number of sequences available from data banks. From the sequences readily available from data banks, there is no indication therefrom as to their potential for diagnostic purposes. For determining good candidates for diagnostic purposes, one could select sequences for either (i) the species-specific or genus-specific detection and identification of commonly encountered bacterial pathogens, (ii) the universal detection of bacterial pathogens from clinical specimens and/or (iii) the specific detection and identification of antibiotic resistance genes.
In our co-pending U.S. patent application (Ser. No. 08/526,840), we described DNA sequences suitable for (i) the species-specific detection and identification of 12 clinically important bacterial pathogens, (ii) the universal detection of bacteria, and (iii) the detection of 17 antibiotic resistance genes. This application described proprietary DNA sequences and DNA sequences selected from data banks (in both cases, fragments of at least 100 base pairs), as well as oligonucleotide probes and amplification primers derived from these sequences. All the nucleic acid sequences described in this patent application enter the composition of diagnostic kits and methods capable of a) detecting the presence of bacteria, b) detecting specifically the presence of 12 bacterial species and 17 antibiotic resistance genes. However, these methods and kits need to be improved since the ideal kit and method should be capable of diagnosing close to 100% of bacterial pathogens and antibiotic resistance genes. For example, infections caused by Enterococcus faecium have become a clinical problem because of its resistance to many antibiotics. Both detection of these bacteria and evaluation of their resistance profiles are desirable. Besides that, novel DNA sequences (probes and primers) capable of recognizing the same and other bacterial pathogens or the same and additional antibiotic resistance genes are also desirable to aim at detecting more target genes and complement our earlier patent application.